Construction of Rat Extracellular Signal-regulated Kinase 1 Gene 3' Untranslated Regions Dual-luciferase Reporter Plasmids and Effect of rno-miR-15b-5p on Its Activitiy
10.3969/j.issn.1006-9771.2017.02.010
- VernacularTitle:大鼠细胞外信号调节激酶1基因3'非编码区双荧光素酶报告载体的构建及rno-miR-15b-5p对其活性的影响
- Author:
Hanjiang LUO
;
Yunfeng XU
;
Xiaoxiao LI
;
Yutao YANG
;
Zhiqing XU
- Publication Type:Journal Article
- Keywords:
rno-miR-15b-5p;
extracellular signal-regulated kinase 1;
overlap polymerase chain reaction;
pmiR-ERK13' UTR;
pmiR-ERK1-mut 3' UTR;
luciferase reporter assay
- From:
Chinese Journal of Rehabilitation Theory and Practice
2017;23(2):166-172
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct dual-luciferase reporter plasmids containing the wild type and mutant rat extracellular signal-regulat-ed kinase 1 (ERK1) gene 3' untranslated regions (UTR) which were used to detect rno-miR-15b-5p's putative target gene. Methods The rat ERK1 gene 3' UTR fragment was amplified by polymerase chain reaction (PCR) from PC12 cell cDNA and cloned into pmiR-RB-ReportTM vector. The mutant rat ERK1 gene 3' UTR fragment was obtained by overlap PCR and inserted into pmiR-RB-ReportTM vector. Successful wild type and mutant recombinant plasmids were confirmed by DNA sequencing. PC12 cells were co-transfected with rno-miR-15b-5p mim-ic and pmiR-ERK13' UTR or pmiR-ERK1-mut 3' UTR and then analyzed by dual-luciferase reporter assay system. The achieved mutation sequence of the target site TGCTGCT was mutated to CGAACGT and GTACACG, respectively. Results The wild-type reporter vector pmiR-ERK13' UTR and the mutant reporter vector pmiR-ERK1-mut 3' UTR were successfully constructed. The rno-miR-15b-5p mimic de-creased the activity of pmiR-ERK13' UTR plasmid (P<0.001) but did not decrease the activity of pmiR-ERK1-mut 3' UTR plasmid. Conclu-sion The recombinant pmiR-ERK13' UTR and pmiR-ERK1-mut 3' UTR plasmids were constructed successfully, and luciferase activities demonstrated that the 3' UTR of ERK1 gene might be a potential target of rno-miR-15b-5p.
